A Salmonella phage RDP-SA-22022 and its continuous production process and application

Abstract

The present invention belongs to the field of microbial technology. The present invention provides a Salmonella phage RDP‑SA‑22022, which has a deposit number of CGMCC NO.45272 and is deposited in the General Microbiology Center of the China National Committee for the Preservation and Administration of Microbiological Cultures on September 21, 2022. The present invention also provides a phage composition. The present invention also provides the application of the Salmonella phage RDP‑SA‑22022 or the phage composition as described above. The present invention also provides a continuous production process of the Salmonella phage RDP‑SA‑22022 as described above. The present invention also provides a preparation method for phage preparations produced by continuous fermentation. The continuous production process of Salmonella phage provided by the present invention, through three-stage series continuous proliferation, not only improves the use efficiency of the equipment, improves the production efficiency, and reduces the production cost, but also enables the phage and the host to evolve together, and the biological characteristics of the phage are greatly improved, which can realize efficient and safe large-scale and continuous production of phages.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and specifically relates to a Salmonella phage RDP-SA-22022 and a continuous production process and application thereof. Background Art

[0002] With the development of the aquaculture industry, large-scale and high-density aquaculture has become a trend in the development of the aquaculture industry. However, the high-density aquaculture environment brings hidden dangers to the occurrence of diseases. Among them, salmonellosis is one of the reasons that seriously affect the development of the aquaculture industry. The use of antibiotics can better prevent and treat salmonellosis and promote the development of the aquaculture industry. However, with the widespread, unreasonable and excessive use of antibiotics, bacteria have gradually developed resistance to antibiotics. For cases of severe clinical infections, antibiotics can no longer play their antibacterial and antibacterial effects, causing serious losses to the aquaculture industry. Bacteriophages are a type of virus that can infect bacteria and are the most common and widely distributed group of viruses. Usually, bacteriophages can be found in places full of bacterial communities, such as farms and animal gastrointestinal tracts. When the disadvantages of excessive use of antibiotics gradually emerge and super-resistant bacteria continue to appear, bacteriophages develop into the prevention of animal diseases, the purification of bacterial diseases in breeding farms and aquaculture, the removal of bacteria in feed and water sources, and food safety and hygiene. The large-scale production of bacteriophages is urgently needed.

[0003] At present, the traditional fermentation production of Salmonella phages has the following problems: First, after each batch of fermentation, the fermentation tank and related pipelines and equipment need to be thoroughly cleaned, disinfected and sterilized. The fermentation equipment cannot be used for actual production during the cleaning and disinfection stage. In addition, when starting a new fermentation batch, it takes some time for the phage host cells to reach the logarithmic growth phase, which reduces the efficiency of equipment use; Second, the above non-productive operations take up a certain amount of time, resulting in low production efficiency. At the same time, long-term batch fermentation may cause the host strain to mutate or produce anti-phage ability, making it impossible for the phage to effectively infect the host cell, which is also a reason for the low production efficiency; Third, in the existing fermentation, all culture media are generally added at one time. As the fermentation process progresses, the phage production capacity decreases in the later stage due to the exhaustion of nutrients or the accumulation of metabolites, resulting in a waste of some raw materials and high production costs. Summary of the invention

[0004] The present invention aims to provide a Salmonella phage RDP-SA-22022 and a continuous production process and application thereof in view of the deficiencies of the prior art. The present invention adopts three-stage series continuous fermentation, which not only improves the use efficiency of equipment, improves production efficiency, and reduces production costs during the continuous fermentation process, but also enables the phage and the host to co-evolve, and the biological characteristics of the phage are greatly improved.

[0005] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0006] In the first aspect, the present invention provides a Salmonella phage RDP-SA-22022, which was obtained from the liver of a sick chicken in a diseased chicken farm in Qingdao, Shandong, and was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on September 21, 2022, with a deposit number of CGMCC No. 45272. The host bacteria used in the experiment of the present invention is Salmonella BS-22024, which was isolated from the liver of a sick chicken in a diseased chicken farm in Qingdao, Shandong, and was preserved together with the Salmonella phage RDP-SA-22022 of the present invention.

[0007] Observation under an electron microscope showed that the Salmonella phage RDP-SA-22022 had a polyhedral, three-dimensionally symmetrical head and a tail connected to the head, the head contained nucleic acid, the head had a diameter of 55 nm, and the tail had a length of 130 nm. According to the classification standards of the International Committee on Taxonomy of Viruses (ICTV), the phage was classified as the family Caudoviridae of the order Caudovirales.

[0008] In a second aspect, the present invention provides a phage composition, comprising the Salmonella phage RDP-SA-22022 as described above.

[0009] In a third aspect, the present invention provides a use of the Salmonella phage RDP-SA-22022 or the phage composition described above in the preparation of at least one of the following products: (1) a product for killing or inhibiting Salmonella; (2) a product for preventing and / or treating poultry diseases caused by Salmonella; (3) a product for preventing and / or treating inflammatory responses caused by Salmonella.

[0010] In a fourth aspect, the present invention provides a continuous production process of the Salmonella phage RDP-SA-22022 as described above, comprising the following steps:

[0011] Step 1: Preparation of seed solution:

[0012] (1) Preparing a Salmonella host bacteria seed solution; preferably, the Salmonella host bacteria is Salmonella BS-22024.

[0013] (2) preparing a Salmonella phage seed solution; preferably, the Salmonella phage is Salmonella phage RDP-SA-22022.

[0014] Step 2: Continuous proliferation of Salmonella phage:

[0015] (1) Prepare the first culture medium in the feed tank A, prepare the second culture medium in the phage seed tank B and the host bacteria seed tank C, adjust the pH, and then sterilize for use; sterilize the phage proliferation tank D1, the phage proliferation tank D2, and the phage proliferation tank D3 and sterilize them for use;

[0016] (2) In the phage seed tank B, the host bacteria seed liquid is inoculated, and after culturing for 4-6 hours, the phage seed liquid is then inoculated, and the phage proliferation liquid is continuously cultured for 36-48 hours to obtain the phage proliferation liquid; preferably, in the phage seed tank B, 2% of the host bacteria seed liquid is inoculated, and after culturing for 4-6 hours, 2% of the phage seed liquid is then inoculated, and the phage proliferation liquid is continuously cultured for 36-48 hours to obtain the phage proliferation liquid;

[0017] (3) After the phage seed tank B is connected to the phage seed liquid for 30-50 hours, the host bacteria seed liquid is connected to the host bacteria seed tank C, and the culture is continued for 4-6 hours to obtain the host bacteria proliferation liquid; preferably, after the phage seed tank B is connected to the phage seed liquid for 40 hours, 2% of the host bacteria seed liquid is connected to the host bacteria seed tank C, and the culture is continued for 4-6 hours to obtain the host bacteria proliferation liquid;

[0018] (4) Add the prepared host bacteria proliferation liquid to the phage proliferation tank D1 at a constant speed; then add the prepared phage proliferation liquid to the phage proliferation tank D1 at the same constant speed; when the proliferation liquid in the phage proliferation tank D1 reaches 70%-80% of the tank volume, transfer the proliferation liquid to the phage proliferation tank D2 at a certain transfer speed; when the proliferation liquid in the phage proliferation tank D2 reaches 70%-80% of the tank volume, transfer the proliferation liquid to the phage proliferation tank D3 at the same transfer speed; when the proliferation liquid in the phage proliferation tank D3 reaches 70%-80% of the tank volume, transfer the final product phage fermentation liquid to the storage tank at the same transfer speed. Preferably, the constant speed is 0.4-0.6% of the total volume of the phage proliferation tank D1 per minute; the transfer speed is 0.8-1.2% of the total volume of the phage proliferation tank D2 or the phage proliferation tank D3 per minute; preferably, the volumes of the phage proliferation tank D1, the phage proliferation tank D2 and the phage proliferation tank D3 are the same.

[0019] Preferably, the specific steps of step (4) in step 2 are as follows: adding the prepared host bacteria proliferation liquid to the phage proliferation tank D1 at a rate of 0.4-0.6% of the total volume of the phage proliferation tank D1 per minute; then adding the prepared phage proliferation liquid to the phage proliferation tank D1 at a rate of 0.4-0.6% of the total volume of the phage proliferation tank D1 per minute; when the proliferation liquid in the phage proliferation tank D1 reaches 70%-80% of the tank volume, then adding the prepared phage proliferation liquid to the phage proliferation tank D2 at a rate of 0.4-0.6% of the total volume of the phage proliferation tank D1 per minute. .8-1.2% of the growth liquid is transferred to the phage growth tank D2, when the growth liquid in the phage growth tank D2 reaches 70%-80% of the tank volume, the growth liquid is transferred to the phage growth tank D3 at a rate of 0.8-1.2% of the total volume of the phage growth tank D3 per minute, when the growth liquid in the phage growth tank D3 reaches 70%-80% of the tank volume, the final product phage fermentation liquid is transferred to the storage tank at a rate of 0.8-1.2% of the total volume of the phage growth tank D3 per minute. More preferably, the constant speed is a speed of 0.6% of the total volume of the phage growth tank D1 per minute; the transfer speed is a speed of 1.2% of the total volume of the phage growth tank D2 or the phage growth tank D3 per minute; preferably, the value-added liquid is transferred when the growth liquid in the phage growth tank D1, the phage growth tank D2 or the phage growth tank D3 reaches 70% of the tank volume.

[0020] As described above, the continuous production process of Salmonella phage RDP-SA-22022, the first culture medium components in the feed tank A in the step 2 are: 2.5% yeast extract, 0.5% glycerol, 0.5% sodium chloride, 0.2% potassium dihydrogen phosphate, 0.2% dipotassium hydrogen phosphate, 20 ppm magnesium chloride, 20 ppm calcium chloride, the above is mass to volume (w / v); pH is 6.0±0.2.

[0021] As described above, the continuous production process of Salmonella phage RDP-SA-22022, the second culture medium components in the phage seed tank B and the host bacteria seed tank C in the step 2 are: 2.5% yeast extract, 0.5% glycerol, 0.5% sodium chloride, 0.2% potassium dihydrogen phosphate, 0.2% dipotassium hydrogen phosphate, 20 ppm magnesium chloride, 20 ppm calcium chloride, the above is mass to volume (w / v); pH is 7.2±0.2.

[0022] As described above, in the continuous production process of Salmonella phage RDP-SA-22022, in the step 2, during the continuous proliferation of the Salmonella phage, according to the respective consumption rates of the second culture medium in the phage seed tank B and the host bacteria seed tank C, the first culture medium is added from the feed tank A at the same speed as their respective consumption rates.

[0023] As described above, the continuous production process of Salmonella phage RDP-SA-22022, the parameters of the phage seed tank B, the host bacteria seed tank C, the phage proliferation tank D1, the phage proliferation tank D2, and the phage proliferation tank D3 in step 2 are: rotation speed of 200-300rpm, temperature of 37°C, ventilation of 1:0.6-0.8vvm; pH of 7.5±0.2.

[0024] In the present invention, when the bacteriophage co-evolves with the host bacteria, it evolves some adaptive strategies to overcome the resistance of the host bacteria and obtain better infection ability. The following is the principle of the co-evolution of the bacteriophage and the host bacteria:

[0025] (1) Evading identification and infection restrictions

[0026] Changing the host bacteria binding site: Phages can evade recognition and binding by host bacteria by changing the structure or diversity of their surface proteins.

[0027] Defense molecules against the host: Some phages can produce antimicrobial peptides to defend against defense molecules produced by host bacteria.

[0028] (2) Increasing the diversity of surface adaptive proteins

[0029] Genome recombination and rearrangement: Bacteriophages have the ability to recombine and rearrange their genomes, which can reassemble or rearrange their genomes to produce new surface protein diversity. Such changes allow phages to evade recognition and attack by host bacteria and increase the success rate of infection.

[0030] Acquiring new infection pathways: Some phages may evolve new infection pathways to adapt to the changing defense strategies of the host bacteria.

[0031] (3) Evolutionary adaptive defense mechanisms

[0032] Resistance to CRISPR-Cas systems: Some phages can evolve the ability to resist the host's CRISPR-Cas system.

[0033] Hiding or subverting the host's defense system: Phages can evolve strategies to hide inside host cells to evade the host's defense system. In addition, they can secrete enzymes to subvert the host cell's defense mechanism.

[0034] These co-evolutions allow phages to adapt to the host bacteria's evolved defenses and maintain their ability to persist infecting them. Host bacteria prevent phage infection by changing phage attachment sites. In addition to changes in bacterial cell walls and flagellar receptors, bacteria have other mechanisms to improve tolerance to phages, such as infection restriction, tolerance gene regulation, horizontal gene transfer, and resistance development.

[0035] During the co-evolution of host bacteria and phages, some properties of phages are improved, so that the phages after continuous proliferation have better biological characteristics and better use value.

[0036] In a fifth aspect, the present invention provides a method for preparing a phage preparation by continuous fermentation, wherein the production process described above is used for production and fermentation, and the final product phage fermentation liquid is post-treated to prepare a phage preparation, and the specific steps are as follows:

[0037] (1) Centrifugation: The proliferated phages are centrifuged in a disc centrifuge at 4000-6000 rpm and a flow rate of ≤100-200 L / H to remove unlysed host and bacterial fragments;

[0038] (2) Use 800nm ​​ceramic membrane to further filter out bacteria and bacterial debris;

[0039] (3) Using 100 kd hollow fiber membrane for concentration and endotoxin removal;

[0040] (4) Filtration sterilization: The concentrated phage is sterilized by three-stage filtration using polypropylene 0.45 μm, polyethersulfone 0.45 μm, and polyethersulfone 0.22 μm;

[0041] (5) adding 6% water-soluble starch, 2% trehalose, 2% pyrrolidone K-30, and 0.5% vitamin C to the proliferation after filtration and sterilization, in the above mass ratios, and concentrating to 1 / 4-1 / 3 of the original volume by falling film concentration at 45-50°C;

[0042] (6) vacuum drying, with the temperature controlled below 45°C and the moisture content after drying being less than 6%, thereby obtaining a bacteriophage preparation.

[0043] In the above-mentioned method for preparing a phage preparation by continuous fermentation, the specific steps of using a hollow fiber membrane for concentration and endotoxin removal in step (3) are as follows: first, the phage value-added liquid is concentrated to 1 / 4-1 / 5 of the original volume using a hollow fiber membrane, and then 8-10 times the volume of purified water is added, and after stirring evenly, the hollow fiber membrane is used to concentrate it to 1 / 4-1 / 5 of the original volume, and this is repeated 2-3 times.

[0044] In the above-mentioned method for preparing a phage preparation by continuous fermentation, the vacuum drying in step (6) is carried out using a vacuum belt dryer.

[0045] The beneficial effects of the present invention are:

[0046] 1. The present invention provides a Salmonella phage RDP-SA-22022, which has good biological properties and does not contain virulence genes and lysogenic genes, enriches phage resources, is of great significance for preventing and controlling diseases caused by Salmonella, and avoids biosafety risks.

[0047] 2. The present invention provides a continuous production process for Salmonella phages, which is achieved through three-stage series connection of a phage proliferation tank D1, a phage proliferation tank D2, and a phage proliferation tank D3, wherein the phage seed tank B flows out the phage proliferation liquid at a constant speed, and replenishes nutrients at the same speed as the consumption speed. The phage proliferation tanks D1, D2, and D3 simultaneously control the pH to ensure the pH required for phage proliferation. The phages and the host evolve together during the continuous culture process, thereby improving the biological characteristics of the phages and realizing efficient and safe large-scale continuous production of phages.

[0048] 3. The continuous production process of Salmonella phage provided by the present invention can reduce the frequency of equipment cleaning, improve the utilization efficiency of equipment, improve production efficiency and reduce production costs during the continuous fermentation process.

[0049] 4. The present invention provides a continuous production process for Salmonella phage. Compared with the traditional batch production process, the phage lysis spectrum is improved from 25 strains out of 31 strains to 28 strains out of 31 strains. This shows that the continuous production process of the present invention broadens the lysis spectrum, can lyse more bacterial species, and broadens the treatment range. In addition, the continuous production process of the present invention makes the 6 lysis spots clearer and brighter, which shows that the process of the present invention improves the lysis quality and the treatment effect, which is of great significance for the development of phage products for preventing and / or treating poultry diseases caused by Salmonella, phage products for killing or inhibiting Salmonella, and phage products for preventing and / or treating inflammatory reactions caused by Salmonella.

[0050] 5. The continuous production process of Salmonella phage provided by the present invention improves the lysis rate compared with the traditional batch production process, can kill bacteria more effectively, improves the bactericidal effect, and has a higher clinical application significance of phage therapy.

[0051] 6. The continuous production process of Salmonella phage provided by the present invention has enhanced phage activity and a longer inhibition time on host bacteria and pathogens compared to the traditional batch production process. This indicates that the co-evolution of phage and host bacteria enables phage to adapt to the evolutionary defense of host bacteria and maintain the ability to continuously infect them, which can inhibit the growth and reproduction of pathogens for a long time and more effectively resist the drug resistance of pathogens. Therefore, the phage after continuous proliferation has better biological characteristics and better application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 This is an electron microscope photo of Salmonella phage RDP-SA-22022;

[0053] Figure 2 A schematic diagram of a continuous production process of Salmonella phage provided by the present invention;

[0054] Figure 3 Lysis curves of Salmonella phage RDP-SA-22022 in batch and continuous production. DETAILED DESCRIPTION

[0055] The following will combine the contents in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0056] In the present invention, unless otherwise specified, the equipment and raw materials used can be purchased from the market or are commonly used in the art. The methods in the following embodiments, unless otherwise specified, are conventional methods in the art. Unless otherwise defined, all technical and scientific terms used in this specification have the same meanings as those commonly understood by those skilled in the art of the present invention.

[0057] Example 1 Isolation and Identification of Salmonella Phage RDP-SA-22022

[0058] 1. Isolation and identification of pathogenic Salmonella BS-22024 (referred to as "BS-22024"):

[0059] Sampling was carried out from a diseased chicken farm in Qingdao, Shandong Province. The liver of the diseased chicken was taken by aseptic operation, and streaked on a selective medium (SS agar medium). After culturing at 37°C for 18-24h, a round, flat, neatly edged, smooth and moist black colony was formed on the medium. The typical colony was picked and streaked for purification for 3 times. Then a single colony was picked and inoculated into 5mL LB broth, and cultured at 37°C and 200rpm for 8h to obtain a uniform turbid bacterial suspension. It was then identified as Salmonella by 16sRNA molecular identification, and further identified as Salmonella by PCR and serum identification. Further PCR examination revealed that the bacterium contained multiple virulence genes, had a strong pathogenic infectivity, and was resistant to multiple antibiotics. It was named BS-22024 according to the laboratory naming rules and stored in a -80°C refrigerator with 20% glycerol.

[0060] 2. Phage isolation and identification:

[0061] (1) Liver treatment: Take 1-2 g of sick chicken liver and place it in a sterilized centrifuge tube. Add 3 steel balls and grind for 2 min. Add 1-2 mL of normal saline and centrifuge at 10,000 rpm for 5 min. Take the supernatant and filter it through a 0.22 μm filter. Collect the filtered supernatant and set aside.

[0062] (2) Preparation of mixed bacterial suspension: 0.2 mL of bacterial suspension and 0.5 mL of filtrate were added to 5 mL of LB broth, shaken and cultured at 37°C, 200 rpm for 2-12 h, then centrifuged at 10,000 rpm for 5 min, the supernatant was filtered through a 0.22 μm filter, and the filtered supernatant was collected for later use;

[0063] (3) Phage isolation: Phages were isolated by double plate method. 0.3 mL of the filtrate of the mixed bacterial suspension and 0.1 mL of the BS-22024 bacterial suspension were mixed evenly, incubated at 37°C for 10 min, and then spread on double plates. After incubation at 37°C for 8-12 h, the results were observed. If there were phages, there would be transparent plaques on the plate. A single plaque was picked up and placed in 1 mL of physiological saline, incubated at 37°C for 10 min, and centrifuged at 10,000 rpm for 5 min. The supernatant was filtered through a 0.22 μm filter. 0.1 mL of the filtrate and 0.2 mL of the bacterial suspension were taken and incubated at 37°C for 10 min, spread on double plates, and incubated at 37°C for 8-12 h for purification. This step was followed for purification 3-4 times until the plaque size was uniform. A phage strain was obtained and named RDP-SA-22022. The diameter of the plaques formed by RDP-SA-22022 on the double plates was 2 mm to 3 mm.

[0064] 3. Phage preservation:

[0065] The phage proliferation liquid was mixed evenly with 40% glycerol at a ratio of 1:1 and then stored in liquid nitrogen.

[0066] Example 2 Electron microscopic observation of Salmonella phage RDP-SA-22022

[0067] 1. Experimental methods:

[0068] Take 20 μL of liquid containing crude phage particles and drop it on the copper mesh. Let it precipitate naturally for 15 minutes, and use filter paper to absorb excess liquid from the side. Add a drop of 2% phosphotungstic acid (PTA) on the copper mesh to stain the phage for 10 minutes, then use filter paper to absorb the staining liquid from the side. After the sample is dry, observe the phage morphology with an electron microscope.

[0069] 2. Experimental results:

[0070] like Figure 1As shown, Salmonella phage RDP-SA-22022 has a polyhedral three-dimensional symmetrical head and a tail connected to the head, the head contains nucleic acid, the head has a diameter of 55nm, and the tail has a length of 130nm. According to the classification standards of the International Committee on Taxonomy of Viruses (ICTV), the phage is classified as the family of the family of the order Caudovirales.

[0071] Example 3 Continuous production process of Salmonella phage RDP-SA-22022

[0072] 1. Preparation of seed solution

[0073] (1) Preparation of host seed solution: a single colony was picked up under a sterile environment and inoculated into 5 mL of LB liquid medium, and cultured at 37°C, 200-300 rpm for 4.0-5.0 h. The cultured host bacteria were inoculated into 100 mL of LB liquid medium at a 3% inoculation amount, and cultured at 37°C, 200 rpm for 4.0-6.0 h. Then, the inoculation amount was inoculated into 1000 mL of LB liquid medium at a 3-4% inoculation amount, and cultured at 37°C, 150-200 rpm for 4.0-5.0 h. After sampling and gradient dilution of the cultured host bacteria seeds, the content was determined by the spread plate method. The content was 6×10 8 -6×10 9 cfu / mL, and placed in a 4℃ refrigerator for later use after the test is qualified.

[0074] (2) Preparation of phage seed solution: Preserved phage seeds (concentration ≥ 6 × 10 6 pfu / mL) and then 100 μL of the mixture was mixed with 100 μL of host bacteria (concentration 6×10 8 -6×10 9 cfu / mE) were placed in the upper culture medium (LB medium + 0.5% agar), poured into double-layer plates, and cultured at 37.0°C for 8.0-12.0 h. The plates with cloudy plaques were selected, and the upper culture medium was washed with 5 mL of LB liquid culture medium, followed by centrifugation at 12000 rpm for 10 min, filtered through a 0.22 μm filter membrane, and the clear liquid was collected.

[0075] The phage seed solution and host seed solution were inoculated into 5 mL of LB medium at a ratio of 3%, and cultured at 37°C, 200 rpm for 4-6 h (cultured until the proliferation solution becomes clear or floccules appear). The cultured phage and host were inoculated into 100 mL of LB liquid medium at a 3% inoculation rate, and cultured at 37°C, 200 rpm for 4-6 h. Then, the cultured phage and host were inoculated into 1000 mL of LB liquid medium at a 3% inoculation rate, and cultured at 37°C, 200 rpm for 4-8 h. The titer of the cultured phage seed solution was determined by the double-layer plate method. The titer was ≥6×108 pfu / mL, and qualified seeds were placed in a 4℃ refrigerator for later use.

[0076] 2. Continuous proliferation of Salmonella phage

[0077] The schematic diagram of the continuous production process of Salmonella phage in this embodiment is as follows Figure 2 shown.

[0078] The steps for the continuous proliferation of Salmonella phage are as follows:

[0079] (1) Prepare the first culture medium in the feed tank A, prepare the second culture medium in the phage seed tank B and the host bacteria seed tank C, adjust the pH as required, and then sterilize at 121°C for 30 minutes for use; sterilize the phage proliferation tank D1, phage proliferation tank D2, and phage proliferation tank D3 and set them aside. The volumes of phage proliferation tank D1, phage proliferation tank D2, and phage proliferation tank D3 are the same.

[0080] (2) In the phage seed tank B, 2% of the host bacteria seed liquid is inoculated, and after culturing for 4 hours, 2% of the phage seed liquid is inoculated and cultured continuously for 48 hours to obtain the phage proliferation liquid. During this period, the host and phage will co-evolve, and the phage performance will be improved.

[0081] (3) After the phage seed tank B is inoculated with the phage seed solution for 40 hours, 2% of the host bacteria seed solution is inoculated into the host bacteria seed tank C and cultured continuously for 4 hours to obtain the host bacteria proliferation solution.

[0082] The prepared host bacteria proliferation liquid is added to the phage proliferation tank D1 at a rate of 0.6% of the total volume of the phage proliferation tank D1 per minute; the prepared phage proliferation liquid is then added to the phage proliferation tank D1 at a rate of 0.6% of the total volume of the phage proliferation tank D1 per minute; when the proliferation liquid in the phage proliferation tank D1 reaches 70% of the tank volume, the proliferation liquid is transferred to the phage proliferation tank D2 at a rate of 1.2% of the total volume of the phage proliferation tank D2 per minute; when the proliferation liquid in the phage proliferation tank D2 reaches 70% of the tank volume, the proliferation liquid is transferred to the phage proliferation tank D3 at a rate of 1.2% of the total volume of the phage proliferation tank D3 per minute; when the proliferation liquid in the phage proliferation tank D3 reaches 70% of the tank volume, the final product phage fermentation liquid is transferred to the storage tank at a rate of 1.2% of the total volume of the phage proliferation tank D3 per minute.

[0083] During the continuous proliferation of Salmonella phage, according to the respective consumption rates of the second culture medium in the phage seed tank B and the host bacteria seed tank C, the first culture medium is supplemented from the feed tank A at the same rate as the respective consumption rates.

[0084] Specifically, the phage proliferation tank D1, the phage proliferation tank D2 and the phage proliferation tank D3 are flow-fed with a polyether silicon defoamer to prevent the generation of foam.

[0085] The parameters of the phage seed tank B, host bacteria seed tank C, phage proliferation tank D1, phage proliferation tank D2 and phage proliferation tank D3 are as follows: the rotation speed is 200-300 rpm, the temperature is 37°C, the ventilation is 1:0.6-0.8 vvm, the gas is compressed air, and the pH is 7.5±0.2.

[0086] The first culture medium in feed tank A is as follows: 2.5% yeast extract powder, 0.5% glycerol, 0.5% sodium chloride, 0.2% potassium dihydrogen phosphate, 0.2% dipotassium hydrogen phosphate, 20 ppm magnesium chloride, 20 ppm calcium chloride, all of which are weight to volume (w / v), and the pH is 6.0±0.2.

[0087] The second culture medium in the phage seed tank B and the host bacteria seed tank C is as follows: 2.5% yeast extract, 0.5% glycerol, 0.5% sodium chloride, 0.2% potassium dihydrogen phosphate, 0.2% dipotassium hydrogen phosphate, 20 ppm magnesium chloride, 20 ppm calcium chloride, all of which are mass to volume (w / v), and the pH is 7.2±0.2.

[0088] 3. Phage post-processing process

[0089] (1) Centrifugation: The proliferated phages are centrifuged in a disc centrifuge at 4000-6000 rpm and a flow rate of ≤100-200 L / H to remove unlysed host and bacterial fragments.

[0090] (2) Use 800nm ​​ceramic membrane to further filter out bacteria and bacterial debris.

[0091] (3) Use 100 kd hollow fiber membrane for concentration and endotoxin removal.

[0092] First, use a hollow fiber membrane to concentrate to 1 / 4-1 / 5 of the original volume, then add 8-10 times the volume of purified water, stir evenly, and use a hollow fiber membrane to concentrate to 1 / 4-1 / 5 of the original volume, and repeat 2-3 times.

[0093] (4) Filtration sterilization: The concentrated phage is sterilized by three-stage filtration using polypropylene 0.45 μm, polyethersulfone 0.45 μm, and polyethersulfone 0.22 μm.

[0094] (4) During the proliferation after filtration and sterilization, 6% water-soluble starch, 2% trehalose, 2% pyrrolidone K-30, and 0.5% vitamin C are added, and falling film concentration is performed at 45-50°C to concentrate the culture to 1 / 4-1 / 3 of the original volume.

[0095] (5) Vacuum drying: Use a belt vacuum machine for drying. The temperature during the drying process is controlled below 45°C, and the moisture content after drying is less than 6%.

[0096] Example 4 Storage stability experiment of samples of belt vacuum drying and freeze drying process

[0097] 1. Experimental methods:

[0098] The difference from Example 3 is that this example uses a freeze dryer to replace belt vacuum drying in the phage post-treatment process.

[0099] The storage stability experiment of samples dried and produced by belt vacuum dryer and freeze dryer is as follows: the samples were placed at 4°C and their activity was measured regularly. The results are shown in Table 1.

[0100] 2. Experimental results:

[0101] As shown in Table 1, after 24 months of storage, the potency of the two samples decreased by two orders of magnitude. Compared with the freeze-drying method, the belt vacuum drying method also had better stability.

[0102] Table 1 Storage stability test results of samples dried in belt vacuum dryer and freeze dryer

[0103]

[0104] Comparative Example 1 Batch Production Process of Salmonella Phage RDP-SA-22022

[0105] 1. Preparation of seed solution

[0106] The steps for preparing the host seed solution and the phage seed solution are the same as those in Example 3 "1. Preparation of seed solution".

[0107] 2. Salmonella phage proliferation:

[0108] The prepared host bacteria seed liquid is inoculated in a seed fermentation tank at a ratio of 3%. After culturing for 3-3.5 hours, when the OD600 value is 0.8-0.9, the host bacteria is inoculated in a proliferation fermentation tank through a transplanting pipe, and the inoculation amount is 3%. The host bacteria is cultured in the proliferation fermentation tank for 3-3.5 hours. When the OD is 0.7-0.9, the phage seed liquid is inoculated with the inoculation amount of the optimal infection multiplicity. After 3 hours of proliferation, the host bacteria with a volume of 8-9% of the proliferation liquid is added in a flow-added manner. The seed age of the host bacteria in the flow-added is 3-6 hours, and the flow-added time is controlled to be 1-2 hours. Then, the culture is continued for 6-8 hours. When the dissolved oxygen rises and the pH tends to be stable, the proliferation is terminated to obtain the phage fermentation liquid.

[0109] In this comparative example, the parameters of the seed tank are: 200-300 rpm, 37°C, ventilation is 1:0.6-0.8 vvm, and the gas is compressed air. The parameters of the fermentation tank are: 120-150 pm, 37°C, ventilation is 1:0.6-0.8 vvm, and the gas is compressed air.

[0110] The culture medium components in the seed fermentation tank and the proliferation fermentation tank are: 2.5% yeast extract powder, 0.5% glycerol, 0.5% sodium chloride, 0.2% potassium dihydrogen phosphate, 0.2% dipotassium hydrogen phosphate, 20 ppm magnesium chloride, 20 ppm calcium chloride, all of which are mass to volume (w / v), pH 7.2.

[0111] Test Example 1 Comparison of phage lysis spectra in continuous and batch production

[0112] 1. Experimental methods:

[0113] 31 strains of Salmonella identified in the laboratory (5 strains of Salmonella typhimurium, 5 strains of Salmonella pullorum, 4 strains of Salmonella Stanley, 1 strain of Salmonella paratyphi A, 1 strain of Salmonella paratyphi B, 1 strain of Salmonella typhi, 7 strains of Salmonella, 1 strain of Salmonella Thompson, 2 strains of Salmonella Sanftenberg, and 4 strains of Salmonella Kentanky) and Salmonella BS-22024 were selected, wherein Salmonella BS-22024 was used as a control, and the phage fermentation broth continuously produced in Example 3 and batch produced in Comparative Example 1 was subjected to a lysis spectrum experiment, and the results are shown in Table 2.

[0114] Table 2 Comparison of lysis spectra of phages produced in batches and continuously

[0115]

[0116]

[0117] Note: + means cleavage is possible, and the cleavage spots are blurred; ++ means cleavage is possible, and the cleavage spots are clear; - means no cleavage

[0118] 2. Experimental results:

[0119] As shown in Table 2, the phages prepared by the two processes, through the lysis spectrum experiment, found that the phage prepared by continuous proliferation has an increased lysis spectrum range, from 25 strains that can be lysed in the original 31 strains to 28 strains that can be lysed in the 31 strains, and the 6 lysis spots of Salmonella typhimurium BS-20031, Stanley Salmonella S182, Stanley Salmonella S191, Salmonella S100, Salmonella pullorum JX37, and Salmonella paratyphi A 50001 become clearer and brighter. It shows that continuous cultivation can increase the lysis spectrum of phages and improve their biological characteristics. In production practice, it can prevent and treat salmonella diseases more effectively.

[0120] Test Example 2 Comparison of phage lysis rates in batch production and continuous production

[0121] 1. Experimental methods:

[0122] Under sterile conditions, 1 mL of phage fermentation broth sample and 1 mL of host bacterial broth (1×10 5 cfu / mL), incubate at 37°C for 15 minutes, mix well and dilute to 10 with saline. -1 -10 -3 Each gradient was plated with 100 μL of each gradient on LB agar plate and incubated at 37°C for 24 hours. Each gradient was repeated twice. At the same time, 1 mL of saline and 1 mL of host bacterial solution (1×10 5 cfu / mL) as a blank control, and repeat the above steps. Select a plate with 30 to 300 colonies for counting. Repeat the test 3 times, take the average value, and the lysis rate is calculated as follows:

[0123] Phage lysis rate = (1-the number of colonies in the treatment group / the number of colonies in the control group) × 100%.

[0124] 2. Experimental results:

[0125] It was calculated that the lysis rate of the Salmonella phage RDP-SA-22022 produced in batches reached 93.63%, while the lysis rate of the continuously produced RDP-SA-22022 on the host was 99.36%, indicating that after continuous cultivation, the phage has undergone beneficial evolution, increased the lysis rate of the host bacteria, and improved the biological performance.

[0126] Test Example 3 Comparison of antibacterial effects of phages produced in batches and continuously

[0127] 1. Experimental methods:

[0128] Phages were added to the host bacterial suspension of BS-22024. Due to the lysis of the host by the phage, the absorbance of OD600 changed, and the lysis curve of the host by the phage was obtained. The host bacterial suspension cultured for 6 hours was inoculated into 100 mL LB medium at a ratio of 2% (V / V), and cultured at 37°C, 200 rpm for 2 hours (OD about 0.60, about 5.0×10 8 cfu / mL), the host bacterial suspension was divided into sterilized conical flasks, and 0.2 mL (titer of about 3.6×10 11pfu / mL, filtered through a 0.22 μm membrane), and the control without phage was used to take the average value. The lysis curves of Salmonella phage RDP-SA-22022 produced in batches and continuously are shown in Figure 3 shown.

[0129] 2. Experimental results:

[0130] Depend on Figure 3 It can be seen that when Salmonella phage is added to the host bacteria BS-22024 bacterial suspension, because the phage has a strong lysis effect on the host, the OD600 absorbance gradually decreases after the phage is connected. As time goes on, the host bacteria that have not been lysed begin to multiply, and the OD600 absorbance gradually increases. Among them, the inhibition time of the phage RDP-SA-22022 fermentation liquid prepared by batch production and fermentation in Comparative Example 1 on the host is 300min, and the inhibition time of the phage RDP-SA-22022 fermentation liquid prepared by continuous production and fermentation in Example 3 on the host is significantly prolonged, from the original 300min to 600min, and the maximum value of OD600 is also reduced. This shows that the activity of the phage propagated by continuous production is enhanced, and the inhibition time of pathogens is prolonged. The enhancement of pathogen lysis and the effective inhibition of growth have very important application value in the treatment of animal diseases.

[0131] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A continuous production process of Salmonella phage RDP-SA-22022, characterized in that: The Salmonella phage RDP-SA-22022 has a deposit number of CGMCC No. 45272 and was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on September 21, 2022, comprising the following steps: Step 1: Preparation of seed solution: (1) Preparing a Salmonella host bacteria seed solution; (2) preparing Salmonella phage seed solution; Step 2: Continuous proliferation of Salmonella phage: (1) Prepare the first culture medium in the feed tank A, prepare the second culture medium in the phage seed tank B and the host bacteria seed tank C, adjust the pH, and then sterilize for use; sterilize the phage proliferation tank D1, the phage proliferation tank D2, and the phage proliferation tank D3 and set them aside; (2) In the phage seed tank B, inoculate 2% of the host bacteria seed solution, culture for 4-6 hours, then inoculate 2% of the phage seed solution, and continue to culture for 36-48 hours to obtain the phage proliferation solution; (3) After the phage seed tank B is inoculated with the phage seed liquid for 30-50 hours, the host bacteria seed liquid is inoculated into the host bacteria seed tank C and cultured continuously for 4-6 hours to obtain the host bacteria proliferation liquid; (4) Add the prepared host bacteria proliferation liquid to the phage proliferation tank D1 at a constant speed; then add the prepared phage proliferation liquid to the phage proliferation tank D1 at the same constant speed; when the proliferation liquid in the phage proliferation tank D1 reaches 70%-80% of the tank volume, transfer the proliferation liquid to the phage proliferation tank D2 at a certain transfer speed; when the proliferation liquid in the phage proliferation tank D2 reaches 70%-80% of the tank volume, transfer the proliferation liquid to the phage proliferation tank D3 at the same transfer speed; when the proliferation liquid in the phage proliferation tank D3 reaches 70%-80% of the tank volume, transfer the final product phage fermentation liquid to the storage tank at the same transfer speed; The specific steps of step (4) in step 2 are as follows: adding the prepared host bacteria proliferation liquid to the phage proliferation tank D1 at a rate of 0.4-0.6% of the total volume of the phage proliferation tank D1 per minute; then adding the prepared phage proliferation liquid to the phage proliferation tank D1 at a rate of 0.4-0.6% of the total volume of the phage proliferation tank D1 per minute; when the proliferation liquid in the phage proliferation tank D1 reaches 70%-80% of the tank volume, then adding the proliferation liquid to the phage proliferation tank D2 at a rate of 0.8% of the total volume of the phage proliferation tank D2 per minute. -1.2% of the growth liquid is transferred to the phage proliferation tank D2, when the growth liquid in the phage proliferation tank D2 reaches 70%-80% of the tank volume, the growth liquid is transferred to the phage proliferation tank D3 at a rate of 0.8-1.2% of the total volume of the phage proliferation tank D3 per minute, when the growth liquid in the phage proliferation tank D3 reaches 70%-80% of the tank volume, the final product phage fermentation liquid is transferred to the storage tank at a rate of 0.8-1.2% of the total volume of the phage proliferation tank D3 per minute; In the step 2, during the continuous proliferation of the Salmonella phage, according to the respective consumption rates of the second culture medium in the phage seed tank B and the host bacteria seed tank C, the first culture medium is added from the feed tank A at the same speed as the respective consumption rates; The parameters of the phage seed tank B, host bacteria seed tank C, phage proliferation tank D1, phage proliferation tank D2, and phage proliferation tank D3 in step 2 are: rotation speed of 200-300 rpm, temperature of 37° C., ventilation of 1:0.6-0.8 vvm; pH of 7.5±0.2; The first culture medium in the feed tank A in step 2 comprises: 2.5% yeast extract, 0.5% glycerol, 0.5% sodium chloride, 0.2% potassium dihydrogen phosphate, 0.2% dipotassium hydrogen phosphate, 20 ppm magnesium chloride, and 20 ppm calcium chloride, where the above are weight to volume (w / v); and the pH is 6.0±0.2; The second culture medium in the phage seed tank B and the host bacteria seed tank C in step 2 comprises: 2.5% yeast extract, 0.5% glycerol, 0.5% sodium chloride, 0.2% potassium dihydrogen phosphate, 0.2% dipotassium hydrogen phosphate, 20 ppm magnesium chloride, and 20 ppm calcium chloride, the above being the mass ratio. volume (w / v); pH 7.2±0.

2.

2. A method for preparing a phage preparation by continuous fermentation, characterized in that: The production process of claim 1 is used for production and fermentation, and the final product phage fermentation liquid is post-treated to prepare a phage preparation, and the specific steps are as follows: (1) Centrifugation: The proliferated phages are centrifuged in a disc centrifuge at 4000-6000 rpm and a flow rate of ≤100-200 L / H to remove unlysed host and bacterial fragments; (2) Use 800nm ​​ceramic membrane to further filter out bacteria and bacterial debris; (3) Using 100 kd hollow fiber membrane for concentration and endotoxin removal; (4) Filtration sterilization: The concentrated phage is sterilized by three-stage filtration using polypropylene 0.45 μm, polyethersulfone 0.45 μm, and polyethersulfone 0.22 μm; (5) Add 6% water-soluble starch, 2% trehalose, 2% pyrrolidone K-30, and 0.5% vitamin C to the proliferation after filtration and sterilization, and use falling film concentration at 45-50°C to concentrate to 1 / 4-1 / 3 of the original volume; (6) Vacuum drying, with the temperature controlled below 45°C and the moisture content after drying being less than 6%, thereby obtaining a bacteriophage preparation.

3. The method for preparing a phage preparation by continuous fermentation according to claim 2, characterized in that: The vacuum drying in step (6) is carried out using a vacuum belt dryer.

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